Throughout this application, various publications are referenced in parenthesis by Author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells. They hold promise for treatment of neurological, psychiatric, and endocrine disorders (De Wied, 1990). Often the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals. Thus, it is expected that the receptors for neuropeptides consist of a large number of members.
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant neuropeptide to be identified in mammalian brain. NPY is an important regulator in both the central and peripheral nervous systems (Heilig et al., 1990) and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and has contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen (Lundberg et al., 1988), intestinal membranes, brain (Hinson et al., 1988), aortic smooth muscle (Mihara et al., 1989), kidney, testis, and placenta (Dumont et al., 1992). In addition, binding sites have been reported in a number of rat and human cell lines (eg. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2), CHP-234, and SMS-MSN)(Aakerlund et al., 1990; Grundemar et al., 1993).
NPY forms a family (called the pancreatic polypeptide family) together with pancreatic polypeptide (PP) and peptide YY (PYY) which all consist of 36 amino acids and have a common tertiary structure, the so-called PP-fold (Glover et al., 1985). Specific features of this family include a polyproline helix in residues 1 through 8, a .beta.-turn in residues 9 through 14, an .alpha.-helix in residues 15 through 30, an outward-projecting C-terminus in residues 30 through 36, and a carboxy terminal amide which appears to be critical for biological activity (Schwartz et al., 1990). The C-terminal amidated residue of these peptides is essential for biological activity (Wahlestedt et al., 1986). Studies with peptide fragments of NPY have indicated that multiple NPY receptor subtypes exist (Wahlestedt et al., 1986). Three major NPY receptor subtypes (Y1, Y2 and Y3) have been defined by pharmacological criteria, with a fourth "atypical" Y1 receptor that has been proposed to regulate feeding behavior. The only NPY receptor which has been cloned to date is the Y1 receptor gene, from mouse (Eva et al., 1992), rat (Eva et al., 1990), and human (Larhammar et al., 1992). One of the key pharmacological features which distinguish Y1 and Y2 is the fact that the Y1 receptor (and not the Y2 receptor) responds to an analog of NPY modified at residues 31 and 34 ([Leu31,Pro34]NPY), whereas the Y2 receptor (and not the Y1 receptor) has high affinity for the NPY peptide carboxyl-terminal fragment NPY-(13-36)(Wahlstedt et al., 1986; Fuhlendorff et al., 1990).
Receptor genes for the other two structurally related peptides, peptide YY (PYY) and pancreatic polypeptide (PP), also have not been cloned. Peptide YY occurs mainly in endocrine cells in the lower gastrointestinal tract (Bottcher et al., 1984). Receptors for PYY were first described in the rat small intestine (Laburthe et al., 1986). This receptor has been defined as PYY-preferring because it displays a 5-10 fold higher affinity for PYY than for NPY (Laburthe et al., 1986; Laburthe, 1990). Recently, a cell line, PKSV-PCT, derived from the proximal tubules of kidneys, has been described to express receptors for PYY (Voisin et al., 1993). Pancreatic polypeptide is predominantly located in endocrine cells of the pancreatic islets (Alumets et al., 1978). PP inhibits pancreatic exocrine secretion and gall bladder contraction (Schwartz, 1983). Interestingly, PP does not appear to be synthesized in or localized to the central nervous system (Di Maggio et al., 1985), but selective PP binding sites have been found in various brain areas, such as the area postrema and adjacent nuclei, regions permeable at the blood-brain barrier (Whitcomb et al., 1990). PP receptors have a much higher affinity for PP than for NPY or PYY (Inui et al., 1990). PP has been shown to bind with high affinity to binding sites on a pheochromocytoma cell line, PC12 (Schwartz et al., 1987). The rank order of affinity for the pharmacologically defined receptors of NPY and related peptides are listed in Table 1.
Using an homology screening approach to clone novel NPY receptor genes, we describe here the isolation and characterization of a novel NPY/PYY/PP receptor clone which we have designated Y4. The Y4 receptor appears to have a unique pharmacological profile, relative to other NPY-related receptors, exhibiting highest affinity for pancreatic polypeptide itself. This receptor clone will enable us to further examine the possibility of receptor diversity and the existence of multiple subtypes within this family of receptors. These could then serve as invaluable tools for drug design for several pathophysiological conditions such as memory loss, depression, anxiety, epilepsy, pain, hypertension, locomotor problems, circadian rhythm disorders, eating/body weight disorders, sexual/reproductive disorders, nasal congestion, diarrhea, gastrointestinal and cardiovascular disorders.